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Environmental and Experimental Botany 67 (2009) 209–214

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

mpact of temperature on olive (Olea europaea L.) pollen performance in relationo relative humidity and genotype

eorgios C. Koubouris a,b,∗, Ioannis T. Metzidakis a, Miltiadis D. Vasilakakis b

Olive Cultivation and Post Harvest Physiology Laboratory, Institute of Olive Tree and Subtropical Plants, National Agricultural Research Foundation (N.AG.RE.F.),grokipio, 73100 Chania, GreecePomology Laboratory, School of Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece

r t i c l e i n f o

rticle history:eceived 20 January 2009eceived in revised form 13 May 2009ccepted 4 June 2009

eywords:liveollen germinationollen tube growthelative humidityemperaturebiotic stress

a b s t r a c t

Olive varieties ‘Koroneiki’, ‘Kalamata’, ‘Mastoidis’ and ‘Amigdalolia’ were employed in two experimentsfor 3 years to assess the effect of temperature on olive pollen germination and tube growth in relation torelative humidity and genotype. Pollen samples were subjected to pre-incubation at 10, 20, 30 or 40 ◦Cin combination with decreased air relative humidity – 80, 40, 30 or 20%, respectively – for 24 h to simu-late temperature stress that is observed during pollen dispersal; and subsequently in vitro cultured. In thesecond experiment, pollen was exposed at 15, 20, 25 and 30 ◦C for 24 h in vitro to evaluate pollen responsein conditions of water and nutrients availability and to determine the optimum pollen germination andtube growth temperatures for each cultivar. The highest pre-incubation temperature treatment (40 ◦C)prevented pollen germination in ‘Koroneiki’ and ‘Mastoidis’, with the less affected varieties (‘Amigdalolia’and ‘Kalamata’) having average germination percentages of only 7.6 and 2%, respectively. Pre-incubationat 30 ◦C had a negative impact on pollen germination in ‘Koroneiki’ (−65%), ‘Kalamata’ (−20%) and‘Amigdalolia’ (−72%) compared to the control (20 ◦C). Pollen pre-incubation at 40 ◦C decreased signif-icantly the pollen tube length in ‘Kalamata’ (−50%) and ‘Amigdalolia’ (−52%). In the second experiment,in vitro pollen germination increased after incubation at 25 ◦C for ‘Koroneiki’ (+6%), ‘Mastoidis’ (+52%),‘Kalamata’ (+10%) and ‘Amigdalolia’ (+10%) compared to the control (20 ◦C). At 30 ◦C germination per-

centages for ‘Mastoidis’, ‘Kalamata’ and ‘Amigdalolia’ were 8, 6 and 14% higher, respectively, compared tothe control (20 ◦C). Pollen tube length also increased with incubation temperature for all of the studiedcultivars. Based on the cumulative stress response index (CSRI) that was calculated for high temperaturestress the varieties were classified: ‘Mastoidis’ and ‘Kalamata’ as tolerant and ‘Koroneiki’ and ‘Amigdalolia’as intermediate at 30 ◦C while all studied cultivars were sensitive at 40 ◦C. The observed strong genotype-differentiated response in high and low temperature stress could be exploited by plant breeders towards

live v

producing new tolerant o

. Introduction

Environmental conditions play a key role for the survival andell being of living organisms. Adverse climatic changes such as the

ncrease of ultraviolet B (UV-B; 280–315 nm) radiation at the Earth’surface (UNEP, 2002) and the predicted temperature increase of

.5–4.5 ◦C (Met Office, 2007) for the near future and up to 6.4 ◦Cill the end of the century (IPCC, 2007) can affect plant physiolog-cal and reproductive processes. Shifts in the extremes of climaticarameters such as temperature and moisture will also have an

∗ Corresponding author at: Olive Cultivation and Post Harvest Physiology Labo-atory, Institute of Olive Tree and Subtropical Plants, National Agricultural Researchoundation (N.AG.RE.F.), Agrokipio, 73100 Chania, Greece. Tel.: +30 28210 83435;ax: +30 28210 93963.

E-mail address: koubourisg@gmail.com (G.C. Koubouris).

098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2009.06.002

arieties.© 2009 Elsevier B.V. All rights reserved.

impact on biodiversity (GBSC, 2007). To which extent temperaturestress will influence plant survival and crops yields is critical inevolutionary, environmental, economic and social terms.

Open field cultivations are more susceptible to abiotic stresssince they are directly exposed at environmental extremes. Inmany plant species such as tomato (Khavari-Nejad, 1980; Peetand Bartholomew, 1996), rice (Baker and Allen, 1993), soybean(Baker et al., 1989) and wheat (Mitchell et al., 1993) optimal tem-perature for reproductive processes is lower than for vegetativegrowth. Additionally, optimum temperature for pollen germinationand tube growth depends on species and varies between cultivars(Loupassaki et al., 1997). Heat stress drastically reduces pollen ger-

mination in bell pepper (Aloni et al., 2001), avocado (Loupassaki etal., 1997), groundnut (Kakani et al., 2002) and tomato (Abdul-Bakiand Stommel, 1995).

Olive (Olea europaea L.) is one of the major fruit tree crops in theMediterranean countries and a headstone for their rural economies

2 nd Experimental Botany 67 (2009) 209–214

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Table 1Monthly means of daily temperature and relative humidity, in Chania area of South-ern Greece, from March to May for the years 2005–2007.

10 G.C. Koubouris et al. / Environmental a

Loumou and Giourga, 2003). Pollination of the olive flower is nec-ssary to achieve a satisfactory fruit yield since parthenocarpicruits are prone to abscission and are of no commercial value. Thus,actors affecting pollen germination could drastically influence pro-uctivity and have serious economic impacts. The high variationbserved in olive flowering expression among years (Ghrisi et al.,999; Lavee et al., 2002) and cultivars (Reale et al., 2006; Seifi et al.,008) establishes the necessity of studying more than one geno-ypes for several years before reaching conclusions regarding theireproductive patterns. These prerequisites are reinforced by theevealed impact of nutrition (Fernandez-Escobar et al., 2008) andiennial bearing (Lavee, 2006) on olive flowering. Determining the

mpact of temperature on flowering of certain cultivars is also nec-ssary in order to assess the potential of olive production losses dueo global warming (De Melo-Abreu et al., 2004).

Previous studies associated temperature with olive bud induc-ion and differentiation (see review paper by Fabbri and Benelli,000), bud development (Bignami et al., 1999), timing of flow-ring (Pérez-López et al., 2008) and fruit set (Ayerza and Coates,004). Yet, the effect of temperature stress on pollen germinationas not been clarified so far. Pinney and Polito (1990) investigatedhe effect of storage temperature (−20 ◦C) on pollen performanceather than the impact of the air temperature during pollination.erri et al. (2008) also focused at storage temperature and reportedhat storage temperatures, regardless of time and genotype fac-ors, had no statistically significant effect on germination rates.ernandez-Escobar et al. (1983) added pistil extracts of differentultivars in the pollen growing medium in order to study pollen-istil incompatibility in relation to temperature. Cuevas et al. (1994)

nvestigated the impact of temperature on pollen germination ofross-pollinated olive plants also incorporating the pollen-pistilncompatibility factor.

Partial dehydration brings the pollen into equilibrium with thenvironment without fatal damage to the cytoplasm (Bassani et al.,994), however uncontrolled water loss induced by high tempera-ure leads to the death of the pollen (Pacini, 1996). Pollen is suppliedith nutrients and water by the paternal plant tissues while it is in

he anther and it is favored by the humidity of the stigmatic surface,n order to survive, as soon as it lands on it. On the contrary, duringollen dispersion which often can last for many days, pollen is sub-

ected to unfavorable temperature and drought conditions that mayffect its germination ability. Herewith, we simulate experimentallyhese two different situations to assess the effect of environmentalactors on pollen performance.

Aim of the present study is to evaluate the effect of temperaturen pollen germination and tube growth. Furthermore, we hypoth-sized that response of the four olive varieties in high and lowemperature stress differs and in order to define genotypic vari-tion we calculated the cumulative stress response index. In ordero assess the interaction of environmental conditions during pollenispersal, temperature stress was applied either prior or during initro germination and the role of relative humidity in reproductiverocesses is also discussed.

. Material and methods

.1. Plant material and site description

Thirty-year-old ‘Koroneiki’, ‘Kalamata’, ‘Mastoidis’ andAmigdalolia’ irrigated olive trees used as pollen donors were

rowing at the Olive Germplasm Collection, Institute of Olive Treesnd Subtropical Plants, N.AG.RE.F., Chania, Greece. Flowering of theour varieties occurs between mid April and mid May depending onach year’s temperature levels during the preceding winter–springeason. Monthly means of daily temperature and relative humidity

March April May

Temperature (◦C) 13.7 15.1 20.0Relative humidity (%) 63.1 59.2 58.9

in Chania area of Southern Greece, from March to May for thethree experimental years 2005–2007, are presented in Table 1.Data were collected from the meteorological station of N.AG.RE.F.,at the Institute of Olive Trees and Subtropical Plants of Chania.

In each year, 10 inflorescences from each of 10 trees of the fourolive varieties were collected at flower growth stages 60–61 (Sanz-Cortes et al., 2002) and pollen was stored at 4 ◦C and used in the next2–3 days. No statistical difference in pollen viability was recordedbetween fresh and stored pollen as measured by the fluoresceindiacetate test (Heslop-Harrison and Heslop-Harrison, 1970) (datanot shown).

2.2. Carry-over effect of heat stress on in vitro pollen performance

In order to assess pollen sensitivity in unfavorable tempera-ture during dispersion and translocation, pollen was incubated at10, 20, 30 or 40 ◦C, for 24 h in a growth chamber (Kottermann,2770, D3162, Hanigsen, Germany). Air relative humidity (RH) wasset at 80, 40, 30 or 20%, respectively; simulating real T/RH com-binations observed in the study area, and was monitored with aThermo-/Hygrometer (TFA, Germany). Subsequently, pollen sam-ples were cultured on solid medium consisting of 0.8% (w/v) agar(Fluka BioChemika, Athens, Greece), 15% (w/v) sucrose (Fluka Bio-Chemika, Athens, Greece), 100 ppm boric acid (Riedel-de Haen,Athens, Greece) and 60 ppm tetracycline hydrochloride (Sigma,Athens, Greece) according to Pinney and Polito (1990) with a slightmodification. Indeed, a micropipette with liquid medium of thesame composition excluding agar used, in order to spread pollenuniformly on the solid medium. Pollen germination and pollentube growth were evaluated after 24 h incubation at 25 ◦C in 16/8 hphotoperiod.

2.3. Role of temperature during in vitro pollen culture

In order to assess the influence of different temperatures inpollen germination of the four olive varieties, pollen samples werein vitro cultured at 15, 20, 25 or 30 ◦C in a growth chamber (Kotter-mann, 2770, D3162, Hanigsen, Germany) on solid medium (Pinneyand Polito, 1990) with the modification described previously. Pollengermination and pollen tube growth were evaluated after 24 h incu-bation in 16/8 h photoperiod.

2.4. Cumulative stress response index (CSRI)

The cumulative stress response index indicates the overall plantresponse in unfavorable environmental conditions and shows therelative sensitivity of different genotypes in a specific stress. A CSRIbased on the concept of Dai et al. (1994), was calculated to evaluatethe overall reproductive response of olive varieties to unfavorableair temperature using the following equation:

CSRI =[

PGt − PGcPGc

+ PTLt − PTLcPTLc

]× 100

where CSRI = cumulative stress response index, PG = pollen ger-mination percentage, PTL = pollen tube length, t = treatment, andc = control.

The temperature of 20 ◦C was considered as the control tem-perature because this is the average temperature for May in the

G.C. Koubouris et al. / Environmental and Experimental Botany 67 (2009) 209–214 211

Fig. 1. Influence of pre-incubation temperature (10, 20, 30 or 40 ◦C, in combinationwith decreased air relative humidity—80, 40, 30 or 20%, respectively) for 24 h on invaMt

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Fig. 2. Influence of pre-incubation temperature (10, 20, 30 or 40 ◦C, in combinationwith decreased air relative humidity—80, 40, 30 or 20%, respectively) for 24 h onpollen tube length for four olive varieties (‘Koroneiki’, ‘Mastoidis’, ‘Kalamata’, and

Fwc

itro pollen germination for four olive varieties (‘Koroneiki’, ‘Mastoidis’, ‘Kalamata’,nd ‘Amigdalolia’). Values are mean of 24 replicates with 50 pollen grains each.eans within a cultivar followed by the same letters do not differ at P < 0.05 (LSD

est).

tudy area (Table 1). Germination and tube length values of there-incubation experiment were employed.

.5. Data recorded and statistical analysis

Pollen germination was evaluated on three fields containingver 50 pollen grains each, in each of four different Petri dishes forach cultivar and treatment. Pollen tube length in vitro was mea-ured for 100 pollen tubes for each cultivar and treatment. In vitroermination experiments were repeated twice resulting in 1200ollen grains observed for germination and 200 for tube length

or each cultivar and treatment. Data were analysed using SPSSSPSS Inc., Chicago, USA) and were subjected to one-way analy-is of variance (ANOVA). The least significant difference (LSD) testst P = 0.05 were used to distinguish treatment differences for theollen germination parameters in the study. The standard errors ofach mean were also calculated. Genotypes were classified based onSRI of pre-incubation temperature as tolerant [>minimum CSRI + 2tandard deviation (S.D.)], intermediate (>minimum CSRI + 1S.D.nd <minimum CSRI + 2S.D.) and sensitive (<minimum TSRI + 1S.D.)ccording to Koti et al. (2005).

. Results

.1. Carry-over effect of heat stress on in vitro pollen performance

Pre-incubation temperature had a marked influence onollen germination in all four genotypes (Fig. 1). The highestre-incubation temperature treatment (40 ◦C) prevented pollenermination in ‘Koroneiki’ and ‘Mastoidis’, with the less affectedarieties (‘Amigdalolia’ and ‘Kalamata’) having average germina-

ig. 3. Image capture for ‘Koroneiki’ pollen following pre-incubation at (A) 40 ◦C and (B)as diminished at the highest temperature not allowing pollen tube length counts to b

ontrol temperature.

‘Amigdalolia’). Values are mean of 200 replicates except for ‘Koroneiki’ and ‘Mas-toidis’ where due to no pollen germination no counts were made and the tube lengthwas considered zero. Means within a cultivar followed by the same letters do notdiffer at P < 0.05 (LSD test).

tion percentages of only 7.6 and 2%, respectively. Pre-incubation at30 ◦C had a negative impact on pollen germination in ‘Koroneiki’(−65%), ‘Kalamata’ (−20%) and ‘Amigdalolia’ (−72%) compared tothe control. On the contrary, a positive effect (+110%), was observedfor ‘Mastoidis’. Pre-incubation at 10 ◦C resulted in germination per-centages ranging between 45 and 60% with negative effect observedfor ‘Kalamata’ (−10%) and ‘Amigdalolia’ (−19%) and positive influ-ence on ‘Koroneiki’ (+14) and ‘Mastoidis’ (+9%), compared to thecontrol.

Pollen pre-incubation at 40 ◦C decreased significantly the pollentube length in ‘Kalamata’ (−50%) and ‘Amigdalolia’ (−52%) (Fig. 2).No counts were made at this treatment for ‘Koroneiki’ and ‘Mas-toidis’ due to the complete pollen germination inhibition (Fig. 3)and tube length was considered to be zero. Compared to the con-trol temperature (20 ◦C), pollen tube length increased at 30 ◦C for‘Mastoidis’ (29%), ‘Kalamata’ (51%) and ‘Amigdalolia’ (27%), exceptfor ‘Koroneiki’ where it was 14% lower. Pre-incubation at 10 ◦C hada positive effect for ‘Koroneiki’, ‘Kalamata’ and ‘Amigdalolia’ whileit reduced tube length of ‘Mastoidis’ pollen grains.

3.2. Role of temperature during in vitro pollen culture

The effect of incubation temperature on in vitro pollen germi-nation of the four olive varieties is shown in Fig. 4. Germinationincreased at 25 ◦C for ‘Koroneiki’ (+6%), ‘Mastoidis’ (+52%), ‘Kala-

mata’ (+10%) and ‘Amigdalolia’ (+10%) compared to the control.At 30 ◦C germination percent for ‘Mastoidis’, ‘Kalamata’ and‘Amigdalolia’ was 8, 6 and 14% higher, respectively, compared tothe control (20 ◦C). However, 14% decreased germination rate wasobserved at the highest temperature for ‘Koroneiki’ pollen. The

20 ◦C, for 24 h and subsequent in vitro culture at 25 ◦C for 24 h. Pollen germinatione implemented. On the contrary, massive pollen germination was observed at the

212 G.C. Koubouris et al. / Environmental and Experimental Botany 67 (2009) 209–214

Fig. 4. Influence of incubation temperature (15, 20, 25 or 30 ◦C, constant rela-t(cd

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Table 2Cumulative stress response index (CSRI) at different levels of pre-incubation tem-perature (10, 30 and 40 ◦C) in comparison to control (20 ◦C) observed for pollengermination and pollen tube length for four olive varieties (‘Koroneiki’, ‘Mastoidis’,‘Kalamata’, and ‘Amigdalolia’).

Treatments Genotypes

Koroneiki Mastoidis Kalamata Amigdalolia

Cumulative stress response index (CSRI)10 ◦C 70 (1) −6.7 (4) 3.3 (3) 33.8 (2)30 ◦C −52.4 (4) 156.4 (1) 50.9 (2) −32.7 (3)40 ◦C −199.6 (3) −200 (4) −137.6 (1) −139.1 (2)

We considered 20 ◦C as the control temperature because this is the average tem-

ive humidity) for 24 h on in vitro pollen germination (%) for four olive varieties‘Koroneiki’, ‘Mastoidis’, ‘Kalamata’, and ‘Amigdalolia’). Values are mean of 24 repli-ates with 50 pollen grains each. Means within a cultivar followed by the same letterso not differ at P < 0.05 (LSD test).

owest germination percentages were counted at 15 ◦C for all thexamined cultivars with average values 30–40% reduced comparedo the controls.

Higher pollen tube lengths were counted at higher tempera-ure levels for all of the studied cultivars (Fig. 5). This trend is clearor ‘Koroneiki’, ‘Mastoidis’ and ‘Amigdalolia’. However, the pictures confusing for ‘Kalamata’ with the lowest (118 �m) and high-st (256 �m) values also observed at 15 and 30 ◦C, respectively,ut with longer pollen tubes counted at 20 ◦C (208 �m) than at5 ◦C (194 �m). The maximum pollen tube lengths were 168 �mor ‘Koroneiki’, 178 �m for ‘Mastoidis’, 256 �m for ‘Kalamata’ and49 �m for ‘Amigdalolia’ and were all observed at 30 ◦C. Similarlyo the germination rates, the minimum pollen tube lengths werell observed at 15 ◦C and they were 10, 16, 43 and 32% shorter for

Koroneiki’, ‘Mastoidis’, ‘Kalamata’ and ‘Amigdalolia’, respectively,omparing to the control (pollen culture at 20 ◦C).

.3. Cumulative stress response index (CSRI)

‘Koroneiki’ and ‘Amigdalolia’ had negative CSRI at both highemperatures (30 and 40 ◦C) while ‘Kalamata’ had negative CSRInly at 40 ◦C (Table 2). In case of ‘Mastoidis’, negative CSRI wasalculated at both temperature extremes (10 and 40 ◦C). Based onSRI for high temperature stress the varieties were classified: ‘Mas-oidis’ and ‘Kalamata’ as tolerant and ‘Koroneiki’ and ‘Amigdalolia’s intermediate at 30 ◦C while all studied cultivars were sensitive at0 ◦C. The genotype classification according to CSRI for low temper-ture stress (10 ◦C) was: ‘Koroneiki’ and ‘Amigdalolia’ as tolerant;

Mastoidis’ and ‘Kalamata’ as intermediate.

. Discussion

In the present paper, we studied the effect of temperature onlive pollen germination and tube growth in relation to relativeumidity and genotype. Short-term exposure at high temperature

ig. 5. Influence of incubation temperature (15, 20, 25 or 30 ◦C, constant relativeumidity) for 24 h on pollen tube length (�m) for four olive varieties (‘Koroneiki’,

Mastoidis’, ‘Kalamata’, and ‘Amigdalolia’). Values are mean of 200 replicates. Meansithin a cultivar followed by the same letters do not differ at P < 0.05 (LSD test).

perature for May in the study area (Table 1). The numbers in parenthesis indicatethe ranks of genotypes given in accordance to their performance in a particularenvironment.

extremes (40 ◦C) combined with low RH (20%) prior in vitro cul-ture had detrimental effect on pollen performance of all studiedcultivars. Our results agree with previous studies in olive and otherplant species. For example, in vitro pollen germination and tubegrowth of ‘Picudo’, ‘Picual’, ‘Manzanila’, ‘Hojiblanca’ and ‘Gordal’were diminished at 35 ◦C (Fernandez-Escobar et al., 1983). Zea mayspollen grains showed a severe decrease in germinability at hightemperature (38 ◦C) stress for 24 h (Herrero and Johnson, 1980) andBrassica napus pollen developing at 35 ◦C had lower germinationrates in vitro (at 23 ◦C) comparing with the control pollen underthe same conditions (Young et al., 2004). Pollen germination wasalso negatively affected by high temperature stress in Glycine maxL. (Koti et al., 2005), Arachis hypogaea L. and Phaseolus vulgaris L.(Prasad et al., 2002, 2003). On the other hand, plant response in tem-perature stress is highly genotype-depended since Brassica juncea,Nicotiana sylvestris and Petunia hybrida pollen was not affected bytemperatures of up to 60 ◦C (Rao et al., 1992, 1995).

A heat shock of 40 ◦C is sufficient to induce mRNAs for heat shockproteins (HSP) in pollen (Hopf et al., 1992) revealing the inducedresistance of plant in order to protect (defence mechanism) the cellsagainst damage and/or death. This temperature proved to be lethalfor the pollen of three of the four olive varieties we studied. Thedetermination of HSP mRNAs presence in the pollen might helpexplain why differential thermotolerance was observed betweenthe four cultivars.

Pre-incubation at 30 ◦C decreased pollen germination for‘Koroneiki’, ‘Kalamata’ and ‘Amigdalolia’ but not for ‘Mastoidis’,confirming the strong genotype–treatment interaction observedin olive (Fernandez-Escobar et al., 1983). Reduced germination at30 ◦C has also been reported for ‘Manzanillo’ (Cuevas et al., 1994).After similar moderate heat stress (32/26 ◦C) there was a substan-tial decrease in Capsicum annuum pollen germination as well as inthe pollen tube growth (Aloni et al., 2001).

Pollen germination decreased after in vitro culture at 30 ◦C sim-ilarly to avocado cultivars (Loupassaki et al., 1997), though, incontrast, tube length further increased. An increase in tempera-ture also reduced pollen germination but accelerated pollen tubegrowth in sweet cherry (Hedhly et al., 2004). ‘Amigdalolia’ pollenshowed improved performance in higher temperatures (25–30 ◦C)when cultured in vitro while it responded relatively well at pre-incubation heat stress (40 ◦C) ranking second out of four cultivarsaccording to the CSRI.

Optimum temperature (20–25 ◦C) for pollen germination ofthe studied cultivars is in agreement with previous studies inolive (Fernandez-Escobar et al., 1983; Cuevas et al., 1994) while‘Amigdalolia’ pollen germinated similarly at 25 and 30 ◦C. Tempera-

tures around 25 ◦C were also the most favorable to accelerate in vivopollen tube growth, to advance fertilization and to obtain a good ini-tial fruit set (Cuevas et al., 1994). Pollen of olive (Fernandez-Escobaret al., 1983), avocado (Loupassaki et al., 1997), pear (Vasilakakis and

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orlingis, 1985), mango (Sukhvibul et al., 2000) and papaya (Cohent al., 1989) cultivars also germinated favorably in vitro around thatemperature.

Pollen culture at 15 ◦C resulted in the lowest germination andube growth rates for all four cultivars. In practice, low temperatureuring flowering could affect fertilization processes, though in vitroollen germination at even lower temperatures (5–10 ◦C) should bevaluated to better comprehend the impacts of low temperaturetress on olive reproductive functions.

Low temperature pre-incubation treatment (10 ◦C) had no con-tant effect on pollen germination while its influence on tube lengthas commonly positive. The temperature we employed is con-

idered a mild low temperature stress approaching average nightemperature for April–May in the study area and therefore norastic pollen degradation should be expected. Pollen vitality wasaintained and subsequent in vitro culture at 25 ◦C resulted in sat-

sfactory germination and tube growth for all four cultivars. Theontrasting influence of low temperature on pollen performancehat was revealed in the two experiments highlights the differ-ntial plant response in abiotic factors in relation to the activehysiological process and plant tissues’ developmental stage. Inpecific, according to our results, low temperature could act pos-tively during dispersal by preventing pollen degradation but it isather undesirable during pollen germination on the stigma sincet could decelerate tube growth through the style of the flower.

Cumulative stress response indices for the four varieties revealheir overall response in low or high temperature stress. Clearlyegative CSRIs at 40 ◦C summarise the detrimental effect of heattress on the pollen characteristics that were analysed previously.ifferentiated CSRIs signs for the 30 ◦C treatment revealed theenotype-specific plant response to abiotic elicitors and discrimi-ated ‘Mastoidis’ and ‘Kalamata’ from ‘Koroneiki’ and ‘Amigdalolia’ollen in two groups: tolerant and intermediate, respectively.inally, ‘Koroneiki’ and ‘Amigdalolia’ responded well at moderatelyow temperature while the impact on the pollen performance ofMastoidis’ and ‘Kalamata’ was neutral. The genotype-differentiatedollen response in temperature stresses provides useful informa-ion regarding germplasm reproductive tissues potential tolerancesowever it does not necessarily extend to the point of classifyingcultivar as tolerant in frost or heat incidents. The impact of tem-erature stresses on physiological processes should also be studiedowards the comprehension of overall plant response to abioticxtremes.

In case that an in vitro study lacks information about the overalllant response and the activation of defence or adaptation mech-nisms it may not fulfil to elucidate the research question thatas initially posed. Our results regarding the marked effect of heat

tress on pollen performance were verified by exposing 3-year-oldKoroneiki’ plants at 36 ± 2 ◦C for 1 day and subsequently collectingnd culturing fresh pollen in vitro at 23 ± 2 ◦C for 24 h (Koubouris etl., unpublished data). A future research goal would be to replicatehe pre-incubation experiment in plant level, employing a wideremperature range as well as it is required detailed investigation on

olecular level in order to understand the mechanisms underlyinghe mode of action of abiotic stresses on pollen grain performance.

. Conclusion

Extreme temperature and relative humidity incidents, even forshort period, reduce pollen germination and growth capacity in

Koroneiki’, ‘Kalamata’, ‘Mastoidis’ and ‘Amigdalolia’ olive and may

ffect fruit set and yield. This result may be of great importance forhoosing the appropriate variety for a certain area according to theicroclimate and to elucidate the correlation between low fruit set

nd incidents of extreme temperature during preceding floweringeriod. The observed strong genotype-differentiated response in

erimental Botany 67 (2009) 209–214 213

high and moderate high temperature stress could be exploited byplant breeders towards producing new heat tolerant olive varieties.Moderately low temperature reduces pollen performance and couldresult in dysfunctions of the fertilization processes given the shortstigma receptivity intervals in most olive varieties.

Acknowledgements

We are grateful to N. Tzortzakis and anonymous reviewers,whose comments led to substantial improvements in this paper.

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